Phospho-Chk2 Activation Kit

The kit contains a primary monoclonal antibody that detects only the phosphorylated form of human Chk2, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.

Chk1 and Chk2 are kinases involved in DNA damage-induced cell-cycle check-point signaling. Chk2 is phosphorylated by ATM kinase in response to DNA damage, and Chk2 activation results in cell-cycle inhibition by p53 phosphorylation and other downstream targets. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is caused by Chk2 autophosphorylation on residues Thr383 and Thr387 in the kinase domain activation loop.

Phosho-Chk2 in A549 cells treated with etoposide was quantitatively assayed using the Phospho-Chk2 Activation Kit, Cellomics ArrayScan HCS Reader and the Cellomics Compartmental Analysis BioApplication Software Module. Induction of DNA damage by etoposide leads to phosphorylation of Chk2 at Thr68 resulting in increased staining of the nucleus. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

 
 
Figure 2. Staining of Phosho-Chk2 in A549 cells treated with vehicle (0.1% DMSO in media) (left) or with 50 µM etoposide for 3 hours (right). Cells were stained according to the kit protocol and imaged using a Cellomics ArrayScan HCS Reader. Dose response curve for phospho-Chk2-stained A549 cells treated with etoposide (bottom). The assay output parameter is the difference in percent cells with phospho-Chk2 staining after etoposide treatment. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of etoposide). EC₅₀ = 1.76 ± 0.05 µM.

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